A screening method for quality markers of aidi preparation

Abstract

The application relates to the technical field of traditional Chinese medicine research, in particular to a screening method for quality markers of Aidi preparation. The method comprises the following steps: firstly, qualitatively analyzing chemical components, then quantitatively analyzing components with high content and capable of quantitative determination in traditional Chinese medicine; investigating the influence of light and temperature factors on the content of each component; according to the compatibility law of the monarch, minister, assistant and guide of the original medicinal materials in the compound, attributing the compounds; evaluating the anti-tumor and anti-angiogenesis activities of the candidate Q-markers and their respective synergistic activities of adriamycin in resisting tumor and anti-angiogenesis and reducing myocardial toxicity. Finally, a seven-dimensional spider web of “compatibility-content-stability-anti-tumor cell activity-synergistic DOX anti-tumor activity-anti-angiogenesis activity-synergistic DOX anti-angiogenesis activity” and a four-dimensional spider web of “compatibility-content-stability-reducing DOX cardiotoxicity” are established, and the Q-markers for resisting tumor and protecting heart toxicity of AD are comprehensively analyzed.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine research technology, specifically a method for screening quality markers for Aidi preparations. Background Technology

[0002] Traditional Chinese medicine (TCM) is an important component of complementary and alternative medicine. It not only helps prevent the development and progression of tumors and enhances the efficacy of chemotherapy drugs, but also alleviates the toxic side effects of chemotherapy, thus improving patients' quality of life. In recent years, an increasing number of clinicians have combined TCM preparations with chemotherapy drugs based on the principle of syndrome differentiation and treatment to achieve the goal of enhancing efficacy and reducing toxicity in tumor treatment. For example, some TCM herbs (such as ginseng, astragalus, Scutellaria barbata, American ginseng, etc.) and some anti-tumor TCM preparations (such as Astragalus Decoction, Aidi Injection, Poria Cocos Injection, Xiaoyaping Injection, Shenqi Fuzheng Injection, Kanglaite Injection, Huachansu Injection, and Brucea javanica Oil Emulsion Injection, etc.) combined with chemotherapy or radiotherapy can improve efficacy and reduce the side effects and complications of chemotherapy and radiotherapy. Therefore, in tumor treatment, the role of TCM preparations in reducing toxicity and anti-tumor effects is equally important.

[0003] Traditional Chinese medicine (TCM) preparations are characterized by "multiple components, multiple targets, and multiple pathways." These characteristics bring a series of limitations to TCM preparations, including unclear material basis in complex chemical systems, uncertain mechanisms of action, and difficulties in achieving effective quality control, which are significant obstacles to the standardization and internationalization of TCM preparations. Furthermore, the quality control of TCM preparations also faces the following problems: ① The specificity of quality control indicators is not strong, failing to adequately represent the role of each herb in the overall formula and failing to reflect the compatibility theory of TCM; ② The correlation between quality indicators and efficacy is weak, lacking evaluation methods that link them to clinical effectiveness; ③ Quality control lacks a holistic perspective, and the development of relevant quality standards is even more challenging for varieties with multiple therapeutic effects. If the quality control of TCM preparations with single therapeutic effects is so challenging, the quality control research for anti-tumor TCM preparations with synergistic and toxicity-reducing effects is even more difficult, and there are currently no reports on the quality control of TCM preparations with two important roles.

[0004] Aidi Injection (AD) is one of the traditional Chinese medicine injections approved by the China Food and Drug Administration, and is also one of the most competitive products in the field of traditional Chinese medicine for treating tumors. AD is a compound traditional Chinese medicine preparation composed of four herbs: blister beetle, ginseng, astragalus, and eleutherococcus senticosus. It has the effects of clearing heat and detoxifying, resolving stagnation and dissipating nodules, and is mainly used for primary liver cancer, lung cancer, rectal cancer, malignant lymphoma, and gynecological malignancies. This product has a definite anti-tumor effect and has been widely used clinically for 28 years, holding a significant market share in the traditional Chinese medicine anti-tumor market.

[0005] Alpha-adrenergic radix (AD) not only possesses anti-tumor effects on its own but also significantly synergistically enhances the anti-tumor effects of chemotherapy drugs while reducing their toxic side effects. Previously, our research group elucidated and clarified, from in vivo and in vitro studies, multiple models, and multiple perspectives, the synergistic anti-tumor effects, synergistic inhibition of angiogenesis, and reduction of DOX cardiotoxicity achieved through the combined use of AD and the chemotherapy drug doxorubicin (DOX). The simultaneous synergistic anti-tumor effect and reduction of chemotherapy drug toxicity by AD is of great significance. This provides important clinical evidence for the combined use of traditional Chinese medicine and chemotherapy drugs in the treatment of tumors, demonstrates the clinical value of traditional Chinese medicine preparations, and reflects the comprehensive therapeutic effects of multi-component, multi-target, and multi-pathway approaches of traditional Chinese medicine compound prescriptions.

[0006] However, as a guarantee of the efficacy and safety of traditional Chinese medicine injections, AD's quality control research is severely insufficient. The drug standard (approval number WS3-B-3809-98) only controls the quality of two components: cantharidin and ginsenoside Re. Clearly, these two simple components cannot represent the combined roles of the four herbs. Studies show that all four herbs in the formula contain anti-tumor components, such as cantharidin in cantharidin, ginsenoside Re, ginsenoside Rb1, and ginsenoside Rg1 in ginseng, astragaloside A and astragaloside II in astragalus, and syringin in eleutherococcus senticosus, all of which have anti-tumor effects. Furthermore, isoflavone glucoside in astragalus has the effect of improving cardiotoxicity, and eleutherococcus senticosus glycoside E and syringin, when used in combination with cantharidin, have a reducing effect on the cardiotoxicity of chemotherapy drugs. Therefore, it is urgent to find scientifically sound, highly specific, comprehensive, and efficacy-related quality markers to improve product standards and promote the healthy development of the pharmaceutical industry.

[0007] The effectiveness of quality markers (Q-markers) in traditional Chinese medicine (TCM) is related to the efficacy of TCM preparations. The strength of their effects directly reflects their contribution to TCM preparations; therefore, examining the "effectiveness" dimension is of great significance. Different TCM preparations have different varieties, dosage forms, routes of administration, and pharmacological effects. Evaluation dimensions for Q-markers should be established based on their own advantages, characteristics, and clinical value.

[0008] Therefore, identifying and establishing a specific seven-dimensional spider web of "compatibility-content-stability-antitumor cell activity-synergistic antitumor activity with DOX-anti-angiogenic activity-synergistic anti-angiogenic activity with DOX" and a four-dimensional spider web of "compatibility-content-stability-reduced DOX cardiotoxicity" to establish a multi-dimensional "spider web" model for identifying Q-markers of the antitumor and cardiotoxicity protective effects of AD is currently a key research direction. Summary of the Invention

[0009] To address the aforementioned technical problems in the prior art, this invention provides a method for screening quality markers for Aidi formulations, comprising the following:

[0010] A method for screening quality markers for Aidi formulations includes the following steps:

[0011] (1) Qualitative analysis of chemical components: The chemical components contained in the preparation were identified by ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap tandem mass spectrometry; Compound Discoverer 3.1 software was used to perform qualitative analysis of the preparation by comparing with the mzCloud, PubChem online database and the local database of high resolution mass spectrometry of Chinese herbal medicine components OTCML, and matching with literature reports.

[0012] (2) Determination of chemical components: The content of target components in the preparation was simultaneously quantitatively determined by UPLC-MS / MS method, and the content of each component was obtained after normalization.

[0013] (3) Compatibility analysis of chemical components: The proportion of each component is assigned according to the roles of principal, assistant, adjuvant, and guide; considering the dose-related relationship, the content ratio of the medicinal materials to which the individual belongs in the prescription is normalized, and the proportion of each component is assigned according to the roles of principal, assistant, adjuvant, and guide. After normalization, the compatibility dimension value of each component is obtained, as shown in formula (3):

[0014]

[0015] Where A is the normalized value of the compatibility dimension of the candidate Q-markers; αi is the proportion of each herb (the principal, assistant, adjuvant, and guide herbs are 4, 3, 2, and 1 respectively); wj is the weight of each herb in the prescription; ∑wj is the total weight of the four herbs.

[0016] Common ingredients are normalized directly according to the content ratio of the herbs they belong to in the prescription, without considering the roles of principal, assistant, adjuvant, and guide herbs.

[0017] (4) Evaluation of the activity of each chemical component: The effect of different concentrations of monomers on the proliferation of corresponding cells was investigated by using the CCK-8 method.

[0018] (5) Evaluation of the synergistic activity of each chemical component with DOX: The CCK-8 method was used to examine the activity of different concentrations of monomers with 1 μmol·L⁻¹. -1 The effect of DOX combined with other drugs on the proliferation of corresponding cells;

[0019] (6) Identification of Q-markers of Aidi formulation based on multidimensional "spider web" model: After normalization of each component, scores of different numerical dimensions are obtained. A spider web diagram is built using Excel 2010, and the comprehensive score is calculated. Based on the comprehensive score, Q-markers of Aidi formulation that exert pharmacological and efficacy effects or safety effects are identified.

[0020] Furthermore, the screening method also includes a stability study of the chemical components: the formulation samples are treated under strong light (4500lx±500lx, 25℃) and high temperature (60℃) conditions for 10 days respectively, and samples are collected at 0 and 10 days respectively to examine the changes in the content of each component.

[0021] A method for screening quality markers of the antitumor activity of an Aidi formulation includes the following steps:

[0022] (1) Qualitative analysis of chemical components: The chemical components contained in the preparation were identified by ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap tandem mass spectrometry; Compound Discoverer 3.1 software was used to perform qualitative analysis of the preparation by comparing with the mzCloud, PubChem online database and the local database of high resolution mass spectrometry of Chinese herbal medicine components OTCML, and matching with literature reports.

[0023] (2) Determination of chemical components: The content of target components in the preparation was simultaneously quantitatively determined by UPLC-MS / MS method, and the content of each component was obtained after normalization.

[0024] (3) Stability study of chemical components: The formulation samples were treated under strong light (4500lx±500lx, 25℃) and high temperature (60℃) for 10 days respectively. Samples were collected on 0 and 10 days respectively to investigate the changes in the content of each component.

[0025] (4) Compatibility analysis of chemical components: The proportion of each component is assigned according to the roles of principal, assistant, adjuvant, and guide; considering the dose-related relationship, the content ratio of the medicinal materials to which the individual belongs in the prescription is normalized, and the proportion of each component is assigned according to the roles of principal, assistant, adjuvant, and guide. After normalization, the compatibility dimension value of each component is obtained, as shown in formula (3):

[0026]

[0027] Where A is the normalized value of the compatibility dimension of the candidate Q-markers; αi is the proportion of each herb (the principal, assistant, adjuvant, and guide herbs are 4, 3, 2, and 1 respectively); wj is the weight of each herb in the prescription; ∑wj is the total weight of the four herbs.

[0028] Common ingredients are normalized directly according to the content ratio of the medicinal materials to which they belong in the prescription, without considering the principal, assistant, adjuvant, and guide herbs.

[0029] (5) Evaluation of the antitumor activity of each chemical component: The effect of different concentrations of monomers on the proliferation of Huh7 cells was investigated using the CCK-8 assay.

[0030] (6) Evaluation of the synergistic antitumor activity of various chemical components with DOX: The CCK-8 assay was used to examine the interaction between different concentrations of monomers and 1 μmol·L⁻¹. -1 Effects of DOX combined with other drugs on Huh7 cell proliferation;

[0031] (7) Identification of Q-markers of Aidi formulation based on multidimensional "spider web" model: After normalization of each component, five dimensions of "compatibility-content-stability-antitumor activity-synergistic DOX antitumor activity" scores are obtained. A spider web diagram is built using Excel 2010, the comprehensive score is calculated, and the Q-markers of Aidi formulation that exert antitumor effect are identified according to the comprehensive score.

[0032] A method for screening quality markers of the cardiotoxic protective effect of an adipoxetine formulation, comprising the following steps:

[0033] (1) Qualitative analysis of chemical components: The chemical components contained in the preparation were identified by ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap tandem mass spectrometry; Compound Discoverer 3.1 software was used to perform qualitative analysis of the preparation by comparing with the mzCloud, PubChem online database and the local database of high resolution mass spectrometry of Chinese herbal medicine components OTCML, and matching with literature reports.

[0034] (2) Determination of chemical components: The content of target components in the preparation was simultaneously quantitatively determined by UPLC-MS / MS method, and the content of each component was obtained after normalization.

[0035] (3) Stability study of chemical components: The formulation samples were treated under strong light (4500lx±500lx, 25℃) and high temperature (60℃) for 10 days respectively. Samples were collected on 0 and 10 days respectively to investigate the changes in the content of each component.

[0036] (4) Compatibility analysis of chemical components: The proportion of each component is assigned according to the roles of principal, assistant, adjuvant, and guide; considering the dose-related relationship, the content ratio of the medicinal materials to which the individual belongs in the prescription is normalized, and the proportion of each component is assigned according to the roles of principal, assistant, adjuvant, and guide. After normalization, the compatibility dimension value of each component is obtained, as shown in formula (3):

[0037]

[0038] Where A is the normalized value of the compatibility dimension of the candidate Q-markers; αi is the proportion of each herb (the principal, assistant, adjuvant, and guide herbs are 4, 3, 2, and 1 respectively); wj is the weight of each herb in the prescription; ∑wj is the total weight of the four herbs.

[0039] Common ingredients are normalized directly according to the content ratio of the medicinal materials to which they belong in the prescription, without considering the principal, assistant, adjuvant, and guide herbs.

[0040] (5) Evaluation of the anti-angiogenic activity of each chemical component: The CCK-8 assay was used, and the IC50 of each monomer against HUVEC cells was calculated using Graphpad Prism 8.0.1. 50 value;

[0041] (6) Evaluation of the synergistic anti-angiogenic activity of each chemical component with DOX: The dimension values ​​of each monomer were obtained by using the CCK-8 method.

[0042] (7) Evaluation of the protective effect of each chemical component against DOX-induced cardiotoxicity: The CCK-8 assay was used to examine the protective effects of different concentrations of the monomer against 1 μmol·L⁻¹ cardiotoxicity. -1 Effects of DOX combined with H9c2 cell proliferation;

[0043] (8) Identification of Q-markers of Aidi formulation based on multidimensional "spider web" model: After normalization of each component, four dimensions of "compatibility-content-stability-reduction of DOX cardiotoxicity" scores are obtained. A spider web diagram is built using Excel 2010, the comprehensive score is calculated, and Q-markers of Aidi formulation that exert cardiotoxicity protection are identified according to the comprehensive score.

[0044] A method for screening quality markers for Aidi formulations includes the following steps:

[0045] (1) Qualitative analysis of chemical components: The chemical components contained in the preparation were identified by ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap tandem mass spectrometry (UPLC-Q-Exactive-Plus-Orbitrap-MS); Compound Discoverer 3.1 software was used to perform qualitative analysis of the preparation by comparing with the mzCloud, PubChem online databases and the local database of high-resolution mass spectrometry of traditional Chinese medicine components OTCML, and by matching with literature reports.

[0046] (2) Determination of chemical components: The content of target components in the preparation was simultaneously quantitatively determined by UPLC-MS / MS method, and the content of each component was obtained after normalization.

[0047] (3) Stability study of chemical components: The formulation samples were treated under strong light (4500lx±500lx, 25℃) and high temperature (60℃) for 10 days respectively. Samples were collected on 0 and 10 days respectively to investigate the changes in the content of each component.

[0048] (4) Compatibility analysis of chemical components: The proportions of each component are assigned according to their roles as principal, assistant, adjuvant, and guide. Considering dose-related factors, the content ratios of the individual medicinal materials in the prescription are normalized. The proportions of each component are assigned according to their roles as principal, assistant, adjuvant, and guide. After normalization, the compatibility dimension values ​​of each component are obtained, as shown in formula (3):

[0049]

[0050] Where A is the normalized value of the compatibility dimension of the candidate Q-markers; αi is the proportion of each herb (the principal, assistant, adjuvant, and guide herbs are 4, 3, 2, and 1 respectively); wj is the weight of each herb in the prescription; and ∑wj is the total weight of the four herbs.

[0051] Common ingredients are normalized directly according to the content ratio of the medicinal materials to which they belong in the prescription, without considering the principal, assistant, adjuvant, and guide herbs.

[0052] (5) Evaluation of the antitumor activity of each chemical component: The effect of different concentrations of monomers on the proliferation of Huh7 cells was investigated using the CCK-8 assay.

[0053] (6) Evaluation of the synergistic antitumor activity of various chemical components with DOX: The CCK-8 assay was used to examine the interaction between different concentrations of monomers and 1 μmol·L⁻¹. -1 Effects of DOX combined with other drugs on Huh7 cell proliferation;

[0054] (7) Evaluation of the anti-angiogenic activity of each chemical component: The method is the same as step (5). The IC50 of each monomer against HUVEC cells was calculated using Graphpad Prism 8.0.1. 50 value;

[0055] (8) Evaluation of the synergistic anti-angiogenic activity of each chemical component with DOX: The method is the same as step (6), and the dimension values ​​of each monomer are obtained;

[0056] (9) Evaluation of the protective effects of each chemical component against DOX-induced cardiotoxicity: The CCK-8 assay was used to examine the effects of different concentrations of the monomer on 1 μmol·L⁻¹ cardiotoxicity. -1 Effects of DOX combined with H9c2 cell proliferation;

[0057] (10) Identification of Q-markers for the antitumor and cardiotoxicity protection of AD based on a multidimensional “spider web” model: After normalization of each component, seven dimensions were obtained: “compatibility-content-stability-antitumor activity-synergistic antitumor activity-antiangiogenic activity-synergistic antiangiogenic activity” and “compatibility-content-stability-reduction of DOX cardiotoxicity”. A spider web diagram was built using Excel 2010, and the comprehensive score was calculated. Based on the comprehensive score, Q-markers for AD to exert antitumor and cardiotoxicity protection were identified.

[0058] Furthermore, the aforementioned Aidi preparation is an Aidi injection made from blister beetle, ginseng, astragalus, and eleutherococcus senticosus.

[0059] Further, in step (2), the target components are: 1. cantharidin; 2. ginsenoside Rg1; 3. ginsenoside Re; 4. ginsenoside Rb1; 5. ginsenoside Rc; 6. ginsenoside Rd; 7. isopyridine; 8. syringin; 9. gentianin; 10. astragaloside A; 11. chlorogenic acid; 12. eleutheroside E; 13. verbascoside isoflavone glucoside; 14. astragaloside II; 15. neochlorogenic acid; 16. cryptochlorogenic acid; 17. eleutheroside E1; 18. ginsenoside F3; 19. ginsenoside Rg2; 20. azelaic acid;

[0060] The chromatographic and mass spectrometric conditions used were as follows: The column was an ACQUITYUPLC BEH C18 1.7 μm, 2.1*50 mm; the mobile phase was: A was 0.2% formic acid-water solution, and B was 0.2% formic acid-acetonitrile solution, with gradient elution (0–1 min, 95% A → 95% A; 1–3 min, 95% A → 10% A; 3–3.5 min, 10% A → 10% A; 3.5–4.5 min, 10% A → 95% A; 4.5–5 min, 95% A → 95% A); the flow rate was 0.35 mL / min. -1 Column temperature 40℃; injection volume 1μL; ion source: electrospray ionization (ESI), capillary voltage 3.5kV, ion source temperature 600℃, solvent gas N2, flow rate 1000L·h -1 Backflush gas N2 (flow rate 150 L·h) -1 ), collision gas Ar (flow rate 0.15 mL·min) -1 The mass spectrometry data acquisition and processing software was MassLynx V4.1 workstation, and the scanning mode was multiple reaction ion monitoring (MRM) with simultaneous detection of positive and negative ions. The detected ions, mass-to-charge ratios, and cone voltages for each component are shown in the table below:

[0061] Detected items ESI Parent ion (m / z) Daughter ions (m / z) Tapered hole voltage (V) Collision voltage (eV) Cantharidin [[M+H] + ]]> 197.1 95.0 25 20 Ginsenoside Rg1 <![CDATA[[M+Na] + ]]> 823.5 643.3 30 35 Ginsenoside Re <![CDATA[[M+Na] + ]]> 969.5 789.6 30 40 Ginsenoside Rb1 <![CDATA[[M+Na] + ]]> 1131.8 365.2 30 55 Ginsenoside Rc <![CDATA[[M-H] - ]]> 1101.5 789.3 40 50 Ginsenoside Rd <![CDATA[[M-H] - ]]> 970.0 790.3 30 40 Isoazine dermatidine <![CDATA[[M+Na] + ]]> 223.0 107.5 30 20 Syringin <![CDATA[[M+Na] + ]]> 395.2 232.0 30 25 Mangbinghuasu <![CDATA[[M-H] - ]]> 269.1 197.0 30 35 Astragaloside A <![CDATA[[M-H] - ]]> 807.3 627.3 30 45 chlorogenic acid <![CDATA[[M-H] - ]]> 353.3 191.1 30 20 Acanthopanax senticosin E <![CDATA[[M+H] + ]]> 765.3 603.0 30 40 Versicolor isoflavone glucoside <![CDATA[[M-H] - ]]> 447.0 285.0 30 15 Astragaloside II <![CDATA[[M-H ]- ]]> 849.5 669.2 30 45 Neochlorogenic acid <![CDATA[[M-H] - ]]> 353.0 191.0 30 30 cryptochlorogenic acid <![CDATA[[M-H] - ]]> 353.2 191.1 30 25 Acanthopanax senticosin E1 <![CDATA[[M+Na] + ]]> 603.0 440.1 30 30 Ginsenoside F3 <![CDATA[[M+Na] + ]]> 794.0 335.0 30 40 Ginsenoside Rg2 <![CDATA[[M+Na] + ]]> 807.5 340.0 30 45 azelaic acid <![CDATA[[M-H] - ]]> 187.0 125.0 35 15 .

[0062] Furthermore, in step (2), the normalization method is performed according to the following formula (1):

[0063]

[0064] Where C is the normalized value of the candidate Q-markers content dimension; The average content of candidate Q-markers in each batch; This represents the maximum average content among candidate Q-markers in each batch.

[0065] Furthermore, the examination of the content changes of each component in step (3) is performed using a normalization method, which is based on the following formula (2):

[0066] Where F is the normalized value of the stability dimension of candidate Q-markers; n represents different conditions (strong light and high temperature); f0 is the content of AD candidate Q-markers on day 0; f h10 The content of AD candidate Q-markers on day 10 under strong light conditions; f t10 The content of AD candidate Q-markers on day 10 under high temperature conditions; F n The percentage change in the content of AD candidate Q-markers under different conditions;

[0067] (1 / F n ) max 1 / F among AD candidate Q-markers n The maximum value in.

[0068] Furthermore, step (5) specifically involves using the CCK-8 assay to examine different concentrations of monomers (final concentrations of 200, 150, 100, 50, 25, and 10 μmol·L⁻¹). -1 Or final concentrations of 100, 50, 25, 5, 1, 0.5, or 0.1 μmol·L⁻¹ -1 Effect of 1 μmol·L on Huh7 cell proliferation -1 DOX and 4% Aidi injection were used as positive control concentrations to evaluate the antitumor activity of 20 quantifiable candidate Q-markers. Each component was assigned a value to obtain a dimension value; cell viability = (A 实验组 -A 空白组 ) / (A 正常组 -A 空白组 )×100%.

[0069] Furthermore, step (6) specifically employs the CCK-8 method to examine different concentrations of monomers (final concentrations of 100, 50, 25, 5, 1, 0.5, and 0.1 μmol·L⁻¹). -1 ) respectively with 1 μmol·L -1 Effect of DOX combined with other drugs on Huh7 cell proliferation, 1 μmol·L -1 DOX was used as a positive control concentration. The concentrations of the above 20 monomers and 1 μmol·L⁻¹ were calculated using the Chou-Talalay combination drug index method. -1 The combined drug effect of DOX is determined by CI<1, which indicates a synergistic effect. The values ​​of each component are then assigned to this dimension.

[0070] Furthermore, step (9) specifically involves using the CCK-8 method to examine different concentrations of monomers (final concentrations of 100, 50, 25, 5, 1, 0.5, and 0.1 μmol·L⁻¹). -1 ) respectively with 1 μmol·L -1 Effect of DOX combined with H9c2 cell proliferation, 1 μmol·L -1 DOX was used as the positive control concentration, and then each component was assigned a value to obtain this dimension value.

[0071] Furthermore, in step (10), the comprehensive score calculation formula is as follows (4):

[0072]

[0073] Where S is the overall score of the candidate Q-marker; α is the angle between two adjacent dimensions; and P is the normalized value of each dimension of the candidate Q-marker.

[0074] Compared with the prior art, the technical effects of this invention are reflected in:

[0075] Taking Aidiinjection (AD) as an example, we comprehensively identified Q-markers for AD's antitumor and cardioprotective effects based on a "spider web" model. First, based on the principle of measurability, qualitative analysis was performed on the chemical components of the formula, followed by quantitative analysis of the components with high content and measurability, serving as measurability evaluation indicators. Based on the principle of stability, the effects of light and temperature on the content of each component in AD were examined, serving as stability evaluation indicators. Based on the principle of compatibility, the compounds were classified according to the principal, assistant, adjuvant, and guide principles of the original medicinal materials in the compound formula. Based on the principle of efficacy, the antitumor and anti-angiogenic activities of candidate Q-markers, as well as their synergistic activities with doxorubicin in antitumor, anti-angiogenic, and cardiotoxicity-reducing effects, were evaluated, serving as efficacy evaluation indicators. A seven-dimensional spider web of "compatibility-content-stability-antitumor cell activity-synergistic antitumor activity with DOX-anti-angiogenic activity-synergistic anti-angiogenic activity with DOX" and a four-dimensional spider web of "compatibility-content-stability-reduced DOX cardiotoxicity" were established to comprehensively identify Q-markers for the antitumor and cardiotoxicity protective effects of adenosine monophosphate (AD). The results identified 12 components as Q-markers for AD. Among them, cantharidin, ginsenoside Re, ginsenoside Rb1, astragaloside II, cryptochlorogenic acid, and ginsenoside Rg2 were Q-markers for AD antitumor activity, while ginsenoside Rd, isopyridine, syringin, eleutheroside E, verrucoside glucoside, and argan acid were Q-markers for AD cardiotoxicity protection. The 12 identified Q-markers comprehensively cover all four herbs in the formula, taking into account both antitumor and cardiotoxicity protective effects. This provides a scientific basis for the quality control of AD and an effective method for identifying comprehensive and rational Q-markers for traditional Chinese medicine preparations with two important functions. Attached Figure Description

[0076] Figure 1 This is the total ion chromatogram of positive and negative ion modes in AD.

[0077] Figure 2 This is a chemical structure diagram of 20 candidate Q-markers in AD.

[0078] Figure 3This refers to the content of 20 candidate Q-markers in 14 batches of AD samples (1. cantharidin; 2. ginsenoside Rg1; 3. ginsenoside Re; 4. ginsenoside Rb1; 5. ginsenoside Rc; 6. ginsenoside Rd; 7. isopyridine; 8. syringin; 9. gentianin; 10. astragaloside A; 11. chlorogenic acid; 12. eleutheroside E; 13. verbascoside; 14. astragaloside II; 15. neochlorogenic acid; 16. cryptochlorogenic acid; 17. eleutheroside E1; 18. ginsenoside F3; 19. ginsenoside Rg2; 20. azelaic acid).

[0079] Figure 4 The stability of 20 candidate Q-markers in 14 batches of AD samples (1. cantharidin; 2. ginsenoside Rg1; 3. ginsenoside Re; 4. ginsenoside Rb1; 5. ginsenoside Rc; 6. ginsenoside Rd; 7. isopyridine; 8. syringin; 9. gentianin; 10. astragaloside A; 11. chlorogenic acid; 12. eleutheroside E; 13. verbascoside; 14. astragaloside II; 15. neochlorogenic acid; 16. cryptochlorogenic acid; 17. eleutheroside E1; 18. ginsenoside F3; 19. ginsenoside Rg2; 20. azelaic acid, compared with the 0d group *P<0.05, **P<0.01, ***P<0.001).

[0080] Figure 5 The IC50 of 20 candidate Q-markers on Huh7 cells 50 value.

[0081] Figure 6 It is the synergistic DOX antitumor activity of 20 candidate Q-markers on Huh7 cells.

[0082] Figure 7 It is the IC50 of 20 candidate Q-markers for HUVEC cells 50 value.

[0083] Figure 8 It is the synergistic DOX anti-angiogenic activity of 20 candidate Q-markers on HUVEC cells.

[0084] Figure 9 The study investigated the effects of 20 candidate Q-markers, when combined with DOX, on the survival rate of H9c2 cells.

[0085] Figure 10 This is a spider web pattern diagram of 20 candidate Q-markers. Detailed Implementation

[0086] The technical solution of the present invention will be further defined below with reference to specific embodiments, but the scope of protection is not limited to the description made.

[0087] Example:

[0088] 1. Materials

[0089] 1.1 Main Instruments

[0090] The main instruments used included a Vanquish UPLC system, a Q Exactive Plus high-resolution mass spectrometer and its matching OTCML local database for high-resolution mass spectrometry of traditional Chinese medicine components (Thermo Fisher Scientific, USA), a triple quadrupole tandem mass spectrometer (Waters Xevo TQS, chromatography workstation: Masslynx 4.0, Waters Corporation, USA), a WD-2A drug stability tester (Beijing Tongde Venture Technology Co., Ltd.), an EL-104 electronic analytical balance (Mettler-Toledo Instruments (Shanghai) Co., Ltd.), an Allegra X-30R Centrifuge centrifuge (Beckman Coulte, USA), an 8000DH carbon dioxide cell incubator (Thermo Fisher Scientific, USA), a TS100 inverted microscope (Nikon Corporation, Japan), a DL-CJ-2N double-person ultra-clean workbench (Beijing Donglian Haer Instrument Manufacturing Co., Ltd.), and a Variskan Lux multi-functional microplate reader (Thermo Fisher Scientific, USA).

[0091] 1.2 Experimental Cells

[0092] The liver cancer cells Huh7, human umbilical vein endothelial cells HUVEC, and rat cardiomyocytes H9c2 were all purchased from the Cell Bank of the Chinese Academy of Sciences in Shanghai.

[0093] 1.3 Medicines and Reagents

[0094] Aidi Injection (10mL / vial, batch numbers 20210630, 20220701, 20220702, 20220703, 20220704, 20220705, 20220706, 20220707, 20220708, 20220709, 20220710, 20220711, 20220712, 20220713, Guizhou Yibai Pharmaceutical Co., Ltd.); Reference Standard Doxorubicin Hydrochloride (batch number...) Product No. J0624B, Dalian Meilun Biotechnology Co., Ltd.; Cantharidin (batch number wkq21042910), Ginsenoside Rg1 (batch number wkq22011001), Ginsenoside Re (batch number wkq21072901), Ginsenoside Rb1 (batch number wkq21122303), Ginsenoside Rc (batch number wkq22051102), Ginsenoside Rd (batch number wkq22052704), Isocyanide (batch number wkq21) 111510), Syringin (batch number wkq22021807), Mangiferin (batch number wkq21061509), Astragaloside A (batch number wkq21072008), Chlorogenic acid (batch number wkq22032106), Acanthopanax senticosin E (batch number wkq22010406), Versicolor isoflavone glucoside (batch number wkq21120603), Astragaloside II (batch number wkq22050713), Neochlorogenic acid (batch number wkq2) 2080406), cryptochlorogenic acid (batch number wkq22072103), eleutheroside E1 (batch number wkq22091503), ginsenoside F3 (batch number wkq22091301), ginsenoside Rg2 (batch number wkq22070502), and azelaic acid (batch number wkq22091403) were all purchased from Victor Biotechnology Co., Ltd. (purity > 98%). Methanol, acetonitrile (Fisher Laboratories, USA), and formic acid were all of analytical grade.

[0095] DMEM medium (lot number 8121448, Gibco, USA); RPMI-1640 medium (lot number 8122742, Gibco, USA); fetal bovine serum (lot number 10091-148, Gibco, USA); CCK-8 kit (lot number GK10001, GLPBIO, USA).

[0096] 2. Methods and Results

[0097] 2.1 Qualitative Analysis of the Chemical Composition of AD

[0098] The chemical components of AD were identified using ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap tandem mass spectrometry (UPLC-Q-Exactive-Plus-Orbitrap-MS). The total ion chromatograms of the AD sample in positive and negative ion modes are shown in [Figure number missing]. Figure 1 The identification results are shown in Table 1. Compound Discoverer 3.1 software was used to perform qualitative analysis of AD by comparing the results with the mzCloud, PubChem online databases, and the OTCML local database of high-resolution mass spectrometry for traditional Chinese medicine components, while also matching with literature reports. A total of 87 chemical components were identified, including 24 triterpenoids, 16 flavonoids, 12 phenylpropanoids, 7 lignans, 6 phenols, 4 amino acids, 3 sugars, 2 coumarins, 1 organic acid, and 12 other compounds.

[0099] Table 1. Component Identification Results of AD

[0100] Tab.1 Identification results of AD chemical constituents

[0101]

[0102]

[0103]

[0104]

[0105]

[0106] 2.2 Determination of AD content

[0107] Chromatographic and mass spectrometric conditions: The chromatographic column was an ACQUITYUPLC BEH C18 1.7μm, 2.1*50mm; the mobile phase was: A was 0.2% formic acid-water solution, and B was 0.2% formic acid-acetonitrile solution, with gradient elution (0–1 min, 95% A → 95% A; 1–3 min, 95% A → 10% A; 3–3.5 min, 10% A → 10% A; 3.5–4.5 min, 10% A → 95% A; 4.5–5 min, 95% A → 95% A); the flow rate was 0.35 mL / min. -1 Column temperature 40℃; injection volume 1μL. Ion source: electrospray ionization (ESI), capillary voltage 3.5kV, ion source temperature 600℃, solvent gas N2, flow rate 1000L·h. -1 Backflush gas N2 (flow rate 150 L·h) -1 ), collision gas Ar (flow rate 0.15 mL·min) -1The mass spectrometry data acquisition and processing software was MassLynx V4.1 workstation. The scanning mode was multiple reaction ion monitoring (MRM) with simultaneous detection of positive and negative ions. The detected ions, mass-to-charge ratios, and cone voltages for each component are shown in Table 2 below. The chemical structures of each component are shown in [Table 2]. Figure 2 .

[0108] The contents of 20 components in AD were simultaneously and quantitatively determined by UPLC-MS / MS. The contents of 14 batches of AD were determined, and the contents of each component were obtained after normalization. The normalization method was as follows (1).

[0109]

[0110] Where C is the normalized value of the candidate Q-markers content dimension; The average content of 14 batches of candidate Q-markers; The maximum value of the average content among 14 batches of candidate Q-markers.

[0111] Table 2 Mass Spectrometry Conditions

[0112] Tab.2 Mass spectrometric conditions

[0113]

[0114]

[0115] The concentrations of the 20 candidate Q-markers ranged from 0.017 to 117.694 μg·mL. -1 ( Figure 3 There were significant differences, with the specific contents as follows: cantharidin 0.536–1.817 μg·mL. -1 Ginsenoside Rg1 0.364~1.083μg·mL -1 Ginsenoside Re: 0.497–0.964 μg·mL -1 Ginsenoside Rb: 10.814–2.082 μg / mL -1 Ginsenoside Rc 0.566~1.527μg·mL -1 Ginsenoside Rd: 0.449–0.998 μg / mL -1 Isocyanide 5.672–15.950 μg·mL -1 Syringin 57.516~89.928μg·mL -1 0.320–0.768 μg·mL of gentianin -1 Astragaloside A: 3.740–9.450 μg / mL-1 Chlorogenic acid 1.783~4.208μg·mL -1 Acanthopanax senticosin E 92.042~117.694μg·mL -1 Verbena isoflavone glucoside 14.487–29.710 μg·mL -1 Astragaloside II 2.301–6.006 μg·mL -1 Neochlorogenic acid 5.352–16.035 μg·mL -1 Cryptochlorogenic acid 10.010~22.099μg·mL -1 Acanthopanax senticosin E 11.063~3.569μg·mL -1 Ginsenoside F3 0.027~0.076μg·mL -1 Ginsenoside Rg2 4.914~12.738μg·mL -1 Azelaic acid 20.845~41.285μg·mL -1 .

[0116] 2.3 Stability Study of AD Components

[0117] AD samples were treated under strong light (4500lx±500lx, 25℃) and high temperature (60℃) conditions for 10 days respectively. Samples were collected on days 0 and 10 to investigate the changes in the content of each component.

[0118] The normalization method is as follows (2).

[0119]

[0120] Where F is the normalized value of the stability dimension of candidate Q-markers; n represents different conditions (strong light and high temperature); f0 is the content of AD candidate Q-markers on day 0; f h10 The content of AD candidate Q-markers on day 10 under strong light conditions; f t10 The content of AD candidate Q-markers on day 10 under high temperature conditions; F n The percentage change in the content of AD candidate Q-markers under different conditions; (1 / F n ) max 1 / F among AD candidate Q-markers n The maximum value in.

[0121] like Figure 4As shown, the stability of seven components in AD—ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1, isozymidine, syringin, astragaloside II, and azelaic acid—is significantly affected by light exposure; while the stability of seven other components—ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1, isozymidine, syringin, gentianin, and verbascoside—is significantly affected by high temperature.

[0122] 2.4 Compatibility Analysis of AD Components

[0123] In the anti-tumor effect, blister beetle is the principal ingredient in the formula, while ginseng, astragalus, and eleutherococcus senticosus are the adjuvant ingredients. In the cardioprotective effect, ginseng, with its effects of greatly replenishing vital energy, restoring pulse and consolidating the body, generating fluids and nourishing blood, tonifying the spleen and lungs, and calming the mind and improving intelligence, enters the spleen, lung, heart, and kidney meridians and is usually the principal ingredient. Astragalus has the effects of tonifying qi and raising yang, consolidating the exterior and stopping sweating, promoting diuresis and reducing edema, and generating fluids and nourishing blood; it also has a tonifying effect similar to ginseng and can enhance the treatment of concurrent symptoms, so it is usually the adjuvant ingredient. Eleutherococcus senticosus has the effects of tonifying qi and strengthening the spleen, tonifying the kidneys and calming the mind; it, along with astragalus, belongs to the category of drugs that enhance the treatment of concurrent symptoms and is also an adjuvant ingredient.

[0124] Eighty-seven chemical components were identified in the AD sample, of which 20 were capable of quantitative analysis. The results of classifying each component of the AD sample into medicinal materials and the properties and flavors of each medicinal material are shown in Table 3.

[0125] The proportions of each component are assigned according to their respective roles as principal, assistant, adjuvant, and guide. Considering dosage correlation, the content ratio of the medicinal materials to which the individual components belong in the prescription is normalized. The proportions of each component are assigned according to their respective roles as principal, assistant, adjuvant, and guide. After normalization, the compatibility dimension values ​​of each component are obtained, as shown in formula (3). Common components are not considered in terms of their roles as principal, assistant, adjuvant, and guide, and are directly normalized according to the content ratio of the medicinal materials to which they belong in the prescription.

[0126]

[0127] Where A is the normalized value of the candidate Q-markers matching dimension; α i The specific gravity of each medicinal ingredient (principal, assistant, adjuvant, and guide herbs are 4, 3, 2, and 1 respectively); w j ∑w represents the weight of each medicinal ingredient in the prescription; j This refers to the total weight of the four medicinal herbs.

[0128] Table 3. Herbal properties and their association with the antitumor and cardiotoxic protective effects of candidate Q-markers in AD.

[0129] Tab.3 Anti-tumour and cardiotoxicity protective effects of candidateQ-markers in AD with attribution of original herbs and chemical properties

[0130]

[0131] 2.5 Evaluation of the antitumor activity of AD components

[0132] The CCK-8 assay was used to investigate different concentrations of the monomer (final concentrations of 200, 150, 100, 50, 25, and 10 μmol·L⁻¹). -1 Or final concentrations of 100, 50, 25, 5, 1, 0.5, or 0.1 μmol·L⁻¹ -1 Effect of 1 μmol·L on Huh7 cell proliferation -1 DOX and 4% AD were used as positive control concentrations to evaluate the antitumor activity of 20 quantifiable candidate Q-markers. Each component was assigned a value to obtain a dimension value. Cell viability = (A 实验组 -A 空白组) / (A 正常组 -A 空白组 )×100%.

[0133] The results are as follows Figure 5 As shown, the IC50 of each monomer on the inhibitory effect of Huh7 cells was calculated using Graphpad Prism 8.0.1. 50 Values, cantharidin, ginsenoside Rb1, ginsenoside Rc, cryptochlorogenic acid, and ginsenoside F3 inhibited the proliferation of Huh7 cells with IC50 values. 50 The values ​​were 1.63, 35.55, 65.46, 83.62 and 63.86 μM, respectively, indicating that the above five compounds have good antitumor activity at a concentration below 100 μM.

[0134] 2.6 Evaluation of the synergistic antitumor activity of AD components and DOX

[0135] The CCK-8 assay was used to investigate the effects of different monomer concentrations (final concentrations of 100, 50, 25, 5, 1, 0.5, and 0.1 μmol·L⁻¹). -1 ) respectively with 1 μmol·L -1 Effect of DOX combined with other drugs on Huh7 cell proliferation, 1 μmol·L -1 DOX was used as a positive control concentration. The concentrations of the above 20 monomers and 1 μmol·L⁻¹ were calculated using the Chou-Talalay combination drug index method.-1 The combined drug effect of DOX was analyzed, where CI < 1 indicates a synergistic effect (the smaller the value, the stronger the synergistic effect). Each component was then assigned a value to this dimension. The results are as follows: Figure 6 As shown, cantharidin, ginsenoside Re, ginsenoside Rb1, ginsenoside Rc, isozymine, syringin, astragaloside A, astragaloside II, neochlorogenic acid, cryptochlorogenic acid, and ginsenoside F3 all exhibit significant synergistic effects with DOX, indicating that these 11 compounds possess good synergistic antitumor activity with DOX at concentrations below 100 μM.

[0136] 2.7 Evaluation of the anti-angiogenic activity of AD components

[0137] The method is the same as under item "2.5". The results are as follows: Figure 7 As shown, the IC50 of each monomer against HUVEC cells was calculated using Graphpad Prism 8.0.1. 50 Values ​​of cantharidin, ginsenoside Re, gentianin, cryptochlorogenic acid, and ginsenoside Rg2 on inhibiting HUVEC cell proliferation. 50 The values ​​were 4.67, 77.87, 78.81, 93.06 and 55.73 μM, respectively, indicating that the above five compounds have good anti-angiogenic activity at a concentration below 100 μM.

[0138] 2.8 Evaluation of the synergistic anti-angiogenic activity of AD components and DOX

[0139] The method is the same as in section "2.6" of this chapter. The results are as follows: Figure 8 As shown, cantharidin, ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1, ginsenoside Rc, isozycin, chlorogenic acid, astragaloside II, neochlorogenic acid, cryptochlorogenic acid, eleutheroside E1, ginsenoside F3, and ginsenoside Rg2 have significant synergistic effects with DOX, indicating that the above 13 compounds have good synergistic anti-angiogenic activity with DOX at concentrations below 100 μM.

[0140] 2.9 Evaluation of the protective effect of AD components against DOX-induced cardiotoxicity

[0141] The CCK-8 assay was used to investigate the effects of different monomer concentrations (final concentrations of 100, 50, 25, 5, 1, 0.5, and 0.1 μmol·L⁻¹). -1 ) respectively with 1 μmol·L -1 Effect of DOX combined with H9c2 cell proliferation, 1 μmol·L -1 DOX was used as the positive control concentration, and then each component was assigned a value to obtain this dimension value. The results are as follows: Figure 9The ginsenosides Rg1, Re, Rd, isopyridine, syringin, gentianin, astragaloside A, chlorogenic acid, eleutheroside E, isoflavone glucoside, eleutheroside E1, Rg2, and azelaic acid shown have the activity of reducing the cardiotoxicity of chemotherapy drugs at 100 μM.

[0142] 2.10 Identification of Q-markers for the antitumor and cardiotoxic protective effects of AD based on a multidimensional "spider web" pattern

[0143] After normalization, seven dimensions were obtained: "Compatibility - Content - Stability - Antitumor Activity - Synergistic Antitumor Activity with DOX - Anti-angiogenic Activity - Synergistic Anti-angiogenic Activity with DOX" and "Compatibility - Content - Stability - Reduced DOX Cardiotoxicity". A spider diagram was created using Excel 2010, and the overall score was calculated. Based on the overall score, Q-markers were identified to determine the antitumor and cardiotoxicity protective effects of AD. Normalized data for each dimension are shown in Tables 4 and 5 below. The results are as follows: Figure 10 As shown. The calculation formula is as follows (4).

[0144]

[0145] Where S is the overall score of the candidate Q-marker; α is the angle between two adjacent dimensions; and P is the normalized value of each dimension of the candidate Q-marker.

[0146] Table 4 Normalized values ​​of the antitumor effects of AD candidate Q-markers

[0147] Tab.4 The standardized data of anti-tumor effect of AD candidate Q-markers

[0148]

[0149]

[0150] Table 5. Normalized data of cardiotoxic protective effects of AD candidate Q-markers

[0151]

[0152] 3 Discussion

[0153] This project establishes a Q-marker evaluation method suitable for the characteristics of Alzheimer's disease (AD) based on the Q-marker concept proposed by Academician Liu Changxiao. First, based on the principle of "measurability," ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap high-resolution mass spectrometry (UPLC-Q-Exactive-Plus-Orbitrap-MS), characterized by high separation efficiency, fast scanning speed, high resolution, and high sensitivity, was used to identify the chemical components in AD. Then, after comparison with available reference standards, ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS), characterized by fast analysis speed, high specificity, high sensitivity, high accuracy, high stability, and simultaneous analysis of multiple indicators, was selected to determine the content of 20 monomeric components. Since traditional Chinese medicine injections differ from oral traditional Chinese medicine preparations, trace components can directly enter the bloodstream with 100% bioavailability, directly exerting pharmacological effects after entering the human body. Therefore, the determination of the content of trace components in AD also requires special attention. While high-performance liquid chromatography (HPLC) is readily applicable for content determination, it cannot adequately address the diverse chemical properties and minute amounts of components in adenosine monophosphate (AD). UPLC-MS / MS, however, solves this problem. Most existing studies employ HPLC to determine the indicator components of a specific herb. Some reports describe the simultaneous determination of five glycosides in AD using RP-HPLC-ELSD, while others use liquid chromatography-tandem mass spectrometry (LC-MS / MS) to determine nine components, but these methods cannot cover components with other chemical properties. This experiment demonstrates that the 20 candidate Q-markers determined using UPLC-MS / MS cover a wide range of content (0.017–117.694 μg·mL⁻¹). -1 The content of cantharidin, ginsenoside Re, ginsenoside Rb1, and ginsenoside F3 was less than 2 μg / mL. -1 These components exhibit good inhibitory effects on tumor cell activity or angiogenesis. Furthermore, since AD ​​is an injectable solution in ampoules, its storage is unaffected by humidity; therefore, only the effects of light and temperature on product stability were investigated. Results showed that under strong light and high temperature conditions, ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1, isozymine, syringin, and chlorogenic acid were chemically unstable. In current standards, ginsenoside Re is one of the important indicators for content determination; therefore, AD should be stored in a cool place.

[0154] After qualitative and quantitative detection of AD, based on the principle of "compatibility," the 20 candidate Q-markers were further classified according to the rules of principal, assistant, adjuvant, and guide combinations. In the anti-tumor effect, cantharides is the principal drug, its main active ingredient being cantharidin, which can induce tumor cell apoptosis and inhibit tumor cell proliferation and angiogenesis. Ginseng is the adjuvant drug, its main active ingredient being ginsenosides. For example, the combination of ginsenoside Rc and cyclophosphamide has a synergistic and toxicity-reducing effect on tumor cells. Astragalus is the adjuvant drug, its main active ingredients being saponins and flavonoids. Astragalus membranaceus inhibits AKT phosphorylation and induces HeLa cell apoptosis in a dose-dependent manner. Furthermore, it can inhibit the growth of xenograft tumors in nude mice. Acanthopanax senticosus is the adjuvant drug, among which eleutheroside E and syringin, when combined with cantharidin, have a synergistic and toxicity-reducing effect. Chlorogenic acid, cryptochlorogenic acid, and neochlorogenic acid are common components in the formula, but since their respective medicinal materials are all "adjuvant drugs," they do not affect the score. In terms of cardioprotective effects, ginseng in the formula has the effects of greatly replenishing vital energy, restoring pulse and consolidating the body, promoting body fluid and nourishing blood, tonifying the spleen and benefiting the lungs, and calming the mind and improving intelligence. It enters the spleen, lung, heart and kidney meridians and is often the principal drug. Astragalus has the effects of tonifying qi and raising yang, consolidating the exterior and stopping sweating, promoting diuresis and reducing swelling, and promoting body fluid and nourishing blood. It has the same tonifying effect as ginseng and can enhance the treatment of concurrent symptoms, so it is an adjuvant drug. For example, isoflavone glucoside, as a representative of isoflavone components in astragalus, has antioxidant, vascular endothelial protection and myocardial improvement effects. Azelaic acid reduces DOX-induced myocardial toxicity by inhibiting BNIP3 (Bcl-2 / E1B-19kDa interacting protein 3)-mediated mitophagy and inhibiting H9c2 cardiomyocyte apoptosis. Acanthopanax senticosus has the effects of tonifying qi and strengthening the spleen, tonifying the kidneys and calming the mind. Among them, eleutheroside E and syringin, when used in combination with cantharidin, have a detoxification effect. It belongs to the category of drugs that enhance the treatment of concurrent symptoms, just like astragalus, and both belong to the category of adjuvant drugs. Since chlorogenic acid, cryptochlorogenic acid, and neochlorogenic acid are not specific components of the medicinal materials in the formula and there is overlap, in the compatibility analysis of AD reducing DOX-induced cardiotoxicity, we first calculate the value of each medicinal material according to the formula in the above compatibility section, and finally take the average value as the dimension value of the compatibility analysis in AD cardioprotective effect.

[0155] The effectiveness of Q-markers is related to the efficacy of traditional Chinese medicine (TCM) preparations, and the strength of their effect directly reflects their contribution to TCM preparations. Therefore, examining the "effectiveness" dimension is of great significance. Previous work by our research group has shown that adjuvant agonists (ADs) can increase DOX blood and intratumoral DOX concentrations by altering the activity or expression of various drug-metabolizing enzymes, thus exerting a "synergistic effect." Experiments have also demonstrated that ADs enhance the antitumor effect of DOX by promoting tumor cell apoptosis (mitochondrial pathway), inhibiting angiogenesis, and suppressing tumor cell proliferation. In vitro cell metabolomics experiments show that ADs enhance the antitumor effect of DOX, possibly by balancing the metabolism of amino acids and energy-related substances; and by reducing the concentration of doxorubicin, a toxic metabolite of DOX, in cardiac tissue and H9c2 cardiomyocytes, thus exerting a "toxicity-reducing effect." The simultaneous synergistic antitumor effect and reduction of chemotherapeutic drug toxicity by ADs is significant. This provides important clinical evidence for the combined use of TCM and chemotherapy drugs in tumor treatment, demonstrates the clinical value of TCM preparations, and reflects the comprehensive therapeutic effects of multi-component, multi-target, and multi-pathway TCM compound treatments. Previous studies have shown that AD alone can inhibit the activity of Huh7 and HUVEC cells, and that its combination with DOX also has a synergistic effect in inhibiting the activity of Huh7 and HUVEC cells and reducing cytotoxicity to H9c2 cells. Therefore, based on the principle of "efficacy," this study evaluated the antitumor and antiangiogenic activities of AD candidate Q-markers by investigating their inhibitory effects on the proliferation of Huh7 and HUVEC cells. Furthermore, the study evaluated the synergistic antitumor and antiangiogenic activities of AD candidate Q-markers with DOX by investigating their inhibition of Huh7, HUVEC, and H9c2 cell activity after combination therapy with AD candidate Q-markers and DOX, and used the Chou-Talalay combination drug index method to calculate the combination drug index of AD candidate Q-markers and DOX.

[0156] Currently, there are no reports, either domestically or internationally, on Q-markers for antitumor traditional Chinese medicine preparations that comprehensively "identify" synergistic and toxicity-reducing effects. Therefore, taking Aldosterone Acid (AD) as an example, this study, based on a "spider web" model, for the first time comprehensively "identifies" Q-markers with two important functions. In the antitumor and cardiotoxicity-protective effects of AD, a comprehensive evaluation of multiple effects was achieved, ultimately selecting six components with high comprehensive scores—cantharidin, ginsenoside Re, ginsenoside Rb1, astragaloside II, cryptochlorogenic acid, and ginsenoside Rg2—as Q-markers for AD's antitumor effects; and six components—eleutheroside E, syringin, ginsenoside Rd, verrucoside glucoside, isopyridine, and argan acid—as Q-markers for AD's cardioprotective effects. Of these 12 Q-markers, cantharidin and ginsenoside Re are content determination indicators in the current AD standard; cantharidin, ginsenoside Re, ginsenoside Rb1, and astragaloside II are four Q-markers that can be used to quantitatively plot drug-time curves and maintain blood drug concentrations for a certain period of time, which to some extent confirms the rationality of the results. Furthermore, the 12 Q-markers selected in this study cover all four medicinal materials in the formula, further demonstrating the comprehensiveness of these 12 Q-markers.

[0157] In summary, the 12 Q-markers identified comprehensively cover all four medicinal herbs in the formula, taking into account both anti-tumor and cardiotoxicity protective effects. This provides a scientific basis for the quality control of Alzheimer's disease (AD) and an effective method for identifying comprehensive and rational Q-markers for traditional Chinese medicine preparations with these two important functions.

[0158] Finally, it should be noted that the above embodiments are merely representative examples of the present invention. Obviously, the technical solution of the present invention is not limited to the above embodiments, and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the content disclosed in this invention should be considered within the scope of protection of this invention.

Claims

1. A method for screening quality markers, antitumor activity markers, and cardiotoxicity protective activity markers for a drug formulation, characterized in that, Includes the following steps: (1) Qualitative analysis of chemical components: The chemical components contained in the preparation were identified by ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap tandem mass spectrometry; Compound Discoverer 3.1 software was used to perform qualitative analysis of the preparation by comparing with the mzCloud, PubChem online database and the local database of high resolution mass spectrometry of Chinese herbal medicine components OTCML, and matching with literature reports. (2) Determination of chemical components: The content of target components in the preparation was simultaneously quantitatively determined by UPLC-MS / MS method, and the content dimension values ​​of each component were obtained after normalization. (3) Stability study of chemical components: The formulation samples were treated for 10 days at 25℃, 4500 lx±500 lx strong light and 60℃ high temperature. Samples were collected on 0 and 10 days to investigate the changes in the content of each component. (4) Compatibility analysis of chemical components: The proportion of each component is assigned according to the roles of principal, assistant, adjuvant, and guide; considering the dose-related relationship, the content ratio of the medicinal materials to which the individual belongs in the prescription is normalized, and the proportion of each component is assigned according to the roles of principal, assistant, adjuvant, and guide. After normalization, the compatibility dimension value of each component is obtained, as shown in formula (3): Where A is the normalized value of the compatibility dimension of the candidate Q-markers; αi is the proportion of each herb, with the principal, assistant, adjuvant, and guide herbs being 4, 3, 2, and 1 respectively; wj is the weight of each herb in the prescription; and ∑wj is the total weight of the four herbs. Common ingredients are normalized directly according to the content ratio of the medicinal materials to which they belong in the prescription, without considering the principal, assistant, adjuvant, and guide herbs. (5) Evaluation of the antitumor activity of each chemical component: The effect of different concentrations of monomers on the proliferation of Huh7 cells was investigated using the CCK-8 assay. (6) Evaluation of the synergistic antitumor activity of various chemical components with DOX: The CCK-8 assay was used to examine the interaction between different concentrations of monomers and 1 μmol·L⁻¹. -1 Effects of DOX combined with other drugs on Huh7 cell proliferation; (7) Evaluation of the anti-angiogenic activity of each chemical component: The method is the same as step (5). The IC50 of each monomer against HUVEC cells was calculated using Graphpad Prism 8.0.

1. 50 value; (8) Evaluation of the synergistic anti-angiogenic activity of each chemical component with DOX: The method is the same as step (6), and the dimension values ​​of each monomer are obtained; (9) Evaluation of the protective effect of each chemical component against DOX-induced cardiotoxicity: The CCK-8 assay was used to examine the effects of different concentrations of the monomer on 1 μmol·L⁻¹ cardiotoxicity. -1 Effects of DOX combined with H9c2 cell proliferation; (10) Identification of Q-markers for the antitumor and cardiotoxicity protection of AD based on the multidimensional "spider web" model: After normalization of each component, seven dimensions of "compatibility-content-stability-antitumor activity-synergistic antitumor activity-antiangiogenic activity-synergistic antiangiogenic activity" and "compatibility-content-stability-reduced cardiotoxicity of DOX" scores and "compatibility-content-stability-reduced cardiotoxicity of DOX" scores were obtained. A spider web diagram was built using Excel 2010, and the comprehensive score was calculated. Based on the comprehensive score, the Q-markers for AD to exert antitumor and cardiotoxicity protection were identified. In step (2), the target components are 1. cantharidin; 2. ginsenoside Rg1; 3. ginsenoside Re; 4. Ginsenoside Rb1; 5. Ginsenoside Rc; 6. Ginsenoside Rd; 7. Isocyanidin; 8. Syringin; 9. Mangiferin; 10. Astragaloside A; 11. Chlorogenic acid; 12. Acanthopanaxside E; 13. Verbena isoflavone glucoside; 14. Astragaloside II; 15. Neochlorogenic acid; 16. Cryptochlorogenic acid; 17. Acanthopanaxside E1; 18. Ginsenoside F3; 19. Ginsenoside Rg2; 20. Azelaic acid; The chromatographic and mass spectrometric conditions used were as follows: The chromatographic column was an ACQUITY UPLC BEH C18 1.7 μm, 2.1*50 mm; the mobile phase was: A was 0.2% formic acid-water solution, and B was 0.2% formic acid-acetonitrile solution, with gradient elution. The specific program was as follows: 0–1 min, 95% A → 95% A; 1–3 min, 95% A → 10% A; 3–3.5 min, 10% A → 10% A; 3.5–4.5 min, 10% A → 95% A; 4.5–5 min, 95% A → 95% A; the flow rate was 0.35 mL / min. -1 Column temperature 40℃; injection volume 1 μL; ion source: electrospray ionization source, capillary voltage 3.5 kV, ion source temperature 600℃, solvent gas N2, flow rate 1000 L·h -1 Backflush gas N2, flow rate 150 L·h -1 Collision gas Ar, flow rate 0.15 mL·min -1 The mass spectrometry data acquisition and processing software was MassLynx V4.1 workstation. The scanning mode was multi-reaction ion monitoring (MRI) with simultaneous detection of positive and negative ions. The detected ions, mass-to-charge ratios, and cone voltages for each component are shown in the table below: 。 2. The screening method for quality markers of Aidi formulation, quality markers of antitumor activity, and quality markers of cardiotoxicity protection according to claim 1, characterized in that, In step (2), the normalization method is as follows: (1) in C Normalized values ​​for the candidate Q-markers content dimension; - C n The average content of candidate Q-markers in each batch; - C max This represents the maximum average content among candidate Q-markers in each batch.

3. The screening method for quality markers of Aidi formulation, quality markers of antitumor activity, and quality markers of cardiotoxicity protection according to claim 1, characterized in that, The change in the content of each component in step (3) is examined using a normalization method, which is based on the following formula (2): in F is the normalized value of the stability dimension of candidate Q-markers; n represents different conditions, strong light and high temperature; f0 is the content of AD candidate Q-markers on day 0; f h10 The content of AD candidate Q-markers on day 10 under strong light conditions; f t10 The content of AD candidate Q-markers on day 10 under high temperature conditions; F n The percentage change in the content of AD candidate Q-markers under different conditions; (1 / F n ) max 1 / of the candidate Q-markers for AD F n The maximum value in.

4. The screening method for quality markers of Aidi formulation, quality markers of antitumor activity, and quality markers of cardiotoxicity protection according to claim 1, characterized in that, Step (5) specifically involves using the CCK-8 assay to examine the effect of different concentrations of single-pair Huh7 cell proliferation, at 1 μmol·L⁻¹. -1 DOX and 4% Aidi injection were used as positive control concentrations to evaluate the antitumor activity of 20 quantifiable candidate Q-markers. Each component was assigned a value to obtain a dimension value; cell viability = (A 实验组 -A 空白组 ) / (A 正常组 -A 空白组 ) × 100%.

5. The screening method for quality markers of Aidi formulation, quality markers of antitumor activity, and quality markers of cardiotoxicity protection according to claim 1, characterized in that, Step (6) specifically involves using the CCK-8 method to examine the interaction of monomers at different concentrations with 1 μmol·L⁻¹. -1 Effect of DOX combined with other drugs on Huh7 cell proliferation, 1 μmol·L -1 DOX was used as a positive control concentration. The concentrations of the above 20 monomers and 1 μmol·L⁻¹ were calculated using the Chou-Talalay combination drug index method. -1 The combined drug effect of DOX is determined by CI<1, which indicates a synergistic effect. Each component is then assigned a value to this dimension.

6. The screening method for quality markers of Aidi formulation, quality markers of antitumor activity, and quality markers of cardiotoxicity protection according to claim 1, characterized in that, Step (9) specifically involves using the CCK-8 method to examine the interaction of monomers of different concentrations with 1 μmol·L⁻¹. -1 Effect of DOX combined with H9c2 cell proliferation, 1 μmol·L -1 DOX was used as the positive control concentration, and then each component was assigned a value to obtain this dimension value.

7. The screening method for quality markers of Aidi formulation, quality markers of antitumor activity, and quality markers of cardiotoxicity protection according to claim 1, characterized in that, The formula for calculating the comprehensive score in step (10) is as follows (4): in S The overall score of the candidate Q-markers; α The angle between two adjacent dimensions; P These are the normalized values ​​for each dimension of the candidate Q-marker.

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